1. Field of the Invention
The present invention relates to adhesive copolymers modeled on bioadhesive proteins secreted by marine organisms. These copolymers are compatible with the metabolism, growth and function of living tissues and/or cells in vitro or in vivo and, consequently, are suitable for use in a wide variety of biomedical applications.
2. Description of Related Art
Polymeric materials have been widely used for implants or other biomedical applications, since they bear close resemblance to natural tissue components such as collagen, which allows direct bonding with other substances. Decades of peptide research have created a wide variety of biomedically useful polypeptides. However, they still are the most underrated and underused polymers considering their impressive properties, which include infusibility, mechanical strength and adhesive capability due to a highly flexible backbone and many functional side chains. Waite, et al., Science, 212:1038-1040 (1981) identified some of nature""s most powerful adhesives, bioadhesive polyphenolic proteins, secreted by marine mussels which live under water and routinely cope with the forces of surf and tides. The naturally-occurring bioadhesive polyphenolic protein is produced and stored in the exocrine phenol gland of the mussel and is deposited onto marine surfaces by the mussel""s foot during the formation of new adhesive plaques. While the natural bioadhesive polyphenolic protein can be extracted and purified according to the procedures set forth in the Journal of Biological Chemistry, 258:2911-2915 (1983) or U.S. Pat. No. 4,496,397, the utility of the natural bioadhesive polyphenolic protein extracted from the mussel is limited by the quantities that can be obtained. Consequently synthetic polymers of these natural bioadhesive proteins have been the focus of a significant amount of research, particularly for their potential use as surgical tissue adhesives.
Decades of investigation into this field has led to the discovery of many different marine organisms which secrete adhesive materials. These organisms include many varieties of mussels, which have different environmental needs and subsequent uses for the adhesives they produce, but are alike in that the materials they use for adhesion and cementing contain many of the same building blocks and apparently operate by the same mechanism. See e.g. J. H. Waite, et al., J. Comp. Physiol. B, 159:517-525 (1989) and L. M. Rzepecki, et al., Mol. Mar. Biol. Biotech., 2:255-279 (1993).
The adhesive proteins isolated from mussels (e.g., Mytilus edulis) have been purified and examined for use as tissue adhesives. Preliminary experiments indicated that these proteins are very effective for formation of adhesive bonds to animal tissues and also exhibit low immunogenicity. See e.g. J. H. Waite, Polym. Prepr., 30(1):181-182 (1990) and C. Saez, et al., Comp. Biochem. Physiol., 98B:569-572 (1991). The major drawbacks with these materials are that (i) their mechanisms of action are poorly understood, (ii) the essential requirements for good adhesion and crosslinking are unknown, (iii) recombinant proteins must be enzymatically treated to generate post-translationally modified residues (i.e. DOPA), and (iv) these proteins cannot be produced inexpensively or in the large quantities necessary for successful commercial application. See e.g. J. H. Waite, Biol. Rev., 58:209-231 (1983) and C. V. Benedict, et al., ACS Symp. Ser., 385:465-483 (1989).
The adhesive precursor proteins have been isolated and sequenced from a wide variety of organisms and are known to show certain characteristics. A partial list of these proteins is given in FIG. 1. Examples of amino acid structures found in adhesive proteins are provided in FIG. 2. It is important to note that these consensus repeats are just that, and that considerable variation is present in the sequence of each protein. The repetitive polypeptides have basic isoelectric points (due to lysine residues), flexible conformations (due to high percentages of small glycine and serine residues), and high levels of the amino acid 3,4-dihydroxyphenyl-L-alanine (DOPA). See e.g. J. H. Waite, et al., Science, 212:1038-1040 (1981) and J. H. Waite, J. Biol. Chem., 258:2911-2915 (1983). The DOPA residues are believed to be primarily responsible for (i) chemisorption of the polymers to surfaces underwater and (ii) covalent cross-linking or setting of the adhesive. J. H. Waite, Comp. Biochem. Physiol., 97B:19 (1990); J. H. Waite, Biol. Bull., 183:178-184 (1992).
A number of groups have studied the bioadhesive qualities of synthetic peptides modeled upon marine adhesive proteins. Yamamoto, et al. report the synthesis of L-DOPA homopolymer as well as copolymers of L-DOPA with L-lysine and L-glutamic acid. H. Yamamoto, et al., Polymer, 19:1115-1117 (1978); H. Yamamoto, et al., Macromolecules, 16:1058-1063 (1983); H. Yamamoto, et al., Biopolymers, 21:1137-1151 (1982); H. Yamamoto, et al. Biopolymers, 18:3067-3076 (1979). They have also reported random L-lysine/L-tyrosine copolymers, and the synthesis of complex random copolypeptides which contain as many as 18 different amino acids, including L-DOPA. H. Yamamoto, et al., Int. J. Biol. Macromol., 12:305-310 (1990); A. Nagai, et al., Bull. Chem. Soc. Japn., 62:2410-2412 (1989); H. Yamamoto, et al., Mar. Chem., 37:131-143 (1992); H. Yamamoto, et al., Mar. Chem., 26:331-338 (1989). Sequentially specific copolymers between L-DOPA and L-lysine or L-glutamic acid were prepared by stepwise condensation procedures. Random copolymers of L-DOPA and L-glutamic acid were prepared by polymerization of N-carboxyanhydride (NCA) monomers. Benedict et. al. at Biopolymers, Inc. have also reported synthetic polymers which were composed of small L-DOPA containing peptides grafted to polyamine backbones. C. B. Benedict, et al., U.S. Pat. No. 4,908,404, Mar. 13, 1990. The materials were reported to form adhesive bonds to a variety of substrates and were found to work well with phosphate buffers.
Studies on a variety of synthetic polypeptides modeled upon marine adhesive proteins indicate that these molecules are promising candidates for use as bioadhesives. H. Yamamoto, J. Chem. Soc. Perkin Trans. I, 613-918 (1987). Additional studies disclose an analysis of adhesive properties of L-lysine/L-tyrosine random copolymers and complex random copolymers where tyrosinase enzyme was used as an oxidizing agent. H. Yamamoto, et al., Int. J. Biol. Macromol., 12:305-310 (1990); A. Nagai, et al., Bull. Chem. Soc. Japn., 62:2410-2412 (1989). These adhesive systems were, however, studied under limited reaction environments such as water and diluted synthetic seawater and were found to form adhesive bonds to iron and Al2O3.
In order to facilitate the commercial applications of synthetic peptides modeled upon marine adhesive proteins, there is a need for methods to precisely control the material aspects of the adhesive matrix. In particular, such controlled manipulation of adhesive polypeptide characteristics such as curing time and adhesive strength have a wide number of applications in different biomedical and related commercial contexts. Unfortunately, while the manipulation of adhesive polypeptide characteristics have a number of biomedical applications, specific methods of controlling the material properties of these molecules have not been disclosed.
To address the need for methods to control the crosslinking of synthetic peptides modeled upon marine adhesive proteins, we disclose methods which allow us to synthesize and manipulate the characteristics of copolypeptides which contain the side-chain functional groups (e.g. catechol and primary amine) that are present in these natural adhesive proteins. Specifically, we examined a range of different copolymers to illustrate the effects of a number of factors on crosslinking behavior including the oxidizing agent, copolypeptide concentration and composition and sequence of the functional groups. By controlling the conditions of the reaction environment under which the peptides are crosslinked, it is possible to optimize a variety of different material characteristics of the crosslinked catechol containing copolypeptide adhesive matrix.
The synthetic adhesives disclosed herein are useful since they are water based and thus do not require the use of hazardous or expensive organic solvents for their application. They are also able to form adhesive bonds to a variety of wet surfaces where nearly all commercial synthetic adhesives fail to form strong bonds. The materials disclosed herein are readily prepared and production can easily be scaled to large quantities, an important consideration when these materials are compared with recombinant or natural marine adhesive proteins which possess similar properties, but which are more expensive. These materials may find use as surgical tissue adhesives, dental adhesives, bone cements, hemostatics, as well as other biomedical and industrial applications involving moisture-resistant adhesion.
This disclosure describes the synthesis of moisture-resistant adhesive polypeptides, conditions for their use, and example applications. Illustrative polypeptides containing the amino acid L-dihydroxyphenylalanine (L-DOPA) were prepared as copolymers with L-lysine, L-glutamic acid, L-serine, L-alanine to give water soluble copolymers. Aqueous solutions of these copolymers, when mixed with various oxidizing agents (including O2, mushroom tyrosinase, Fe3+, H2O2, ROOR, and IO4xe2x88x92), formed crossirinked networks which were found to form moisture-resistant adhesive bonds to a variety of substrates (including aluminum, Al2O3, iron, glass, wood, and plastics). The novel features of this system are that the adhesive components are water-based, the polymers are derived from biological sources and may be biocompatible/biometabolizable, the polymers show exceptional bonding capabilities toward wet materials including biological tissues, and the copolymers can be readily prepared in large quantities. The methods disclosed herein are represented by a number of embodiments. One embodiment provides a method of crosslinking a catechol containing polypeptide including the steps of exposing the catechol containing polypeptide to a reaction environment and controlling a condition within the reaction environment to modulate a characteristic of the crosslinked catechol containing polypeptide. In a variation of this embodiment a second condition within the reaction environment is controlled to further modulate a characteristic of the crosslinked catechol containing polypeptide. In one embodiment of these methods, the catechol containing polypeptide is a synthetic polypeptide.
In a typical embodiment of these methods, the characteristic of the crosslinked catechol containing polypeptide is modulated by controlling a factor within the reaction environment selected from the group consisting of oxidizing agent, pH, temperature and polymerization time. In a preferred embodiment, the crosslinked catechol containing polypeptide is modulated by controlling an oxidizing agent, which can be an enzyme like mushroom tyrosinase, or an inorganic chemical, such as O2, H2O2, NaIO4 and Fe(H2O)63+. In yet another embodiment, the characteristic of the crosslinked catechol containing polypeptide is modulated by controlling a factor within the reaction environment selected from the group consisting of polypeptide monomer composition, polypeptide molecular weight, and polypeptide concentration.
In preferred embodiments, the characteristic of the crosslinked catechol containing polypeptide that is modulated is selected from the group consisting of adhesive strength, gellation time and viscosity. In an illustrative embodiment of these methods, the adhesive strength is modulated by controlling the substrate with which the crosslinked catechol containing polypeptide interacts, for example aluminum, steel or glass.
In related embodiments, the invention disclosed herein provides methods of modulating a characteristic of a crosslinked catechol containing polypeptide including modulating at least one factor within the crosslinking reaction environment, wherein the characteristic is selected from the group consisting of adhesive strength, gellation time and viscosity. In a typical embodiment of a method directed to modulating adhesive strength, the factor within the crosslinking reaction environment that modulates adhesive strength is selected from the group consisting of pH, temperature, oxidizing agent, curing time, polypeptide monomer composition, polypeptide molecular weight, polypeptide concentration and substrate with which the crosslinked catechol containing polypeptide interacts. In a typical embodiment of a method directed to modulating viscosity or gellation time or both viscosity and gellation time, the factor within the crosslinking reaction environment that modulates these characteristics is either the oxidizing agent utilized in the crosslinking reaction, the pH or both oxidizing agent and the pH.
By utilizing synthetic polymers, our disclosure can avoid the complexities associated with synthesis and analysis of natural proteins by use of peptides and polymers which contain only the minimal functional groups necessary for cement formation. Concurrent polymer synthesis coupled with characterization has lead to a rapid convergence of crosslink mechanism determination and discovery of the essential adhesive components. Simplified materials also allow the design and synthesis of marketable adhesives prepared through low cost, high volume polymerization techniques.
Using known compositions of many natural adhesive proteins, we prepared sequentially random copolypeptides through copolymerization of a few select xcex1-amino acid N-carboxy anhydrides (NCAs). H. R. Kricheldorf, xcex1-Amino acid-N-Carboxyanhydrides and Related Heterocycles, Springer-Verlag, New York, (1987). NCAs are readily prepared from amino acids by phosgenation and can be polymerized into high molecular weight polypeptides via successive ring opening addition reactions which liberate carbon dioxide. A similar approach for making adhesive polymers using NCA polymerizations has been reported by Yamamoto and coworkers. A. Nagai, et al., Bull. Chem. Soc. Japn., 62:2410-2412 (1989); H. Yamamoto, et al., Int. J. Biol. Macromol., 12:305-310 (1990); H. Yamamoto, J. Chem. Soc. Perkin Trans. I, 613-918 (1987). They have prepared random copolypeptides containing the amino acids Glutamic acid, DOPA, Tyrosine, and Lysine, as well as a complex mixture of 17 different NCAs. H. Yamamoto, et al., Mar. Chem., 26:331-338 (1989); H. Yamamoto, et al., Mar. Chem., 37:131-143 (1992). Their work was focused on conformational analyses of the polymers and there was no attempt to determine the amount of moisture resistance in adhesion measurements. Furthermore, the adhesive roles of the catechol versus the oxidized o-quinone functionalities of DOPA were not separately evaluated.
We disclose methods of manipulating simple catechol containing copolypeptides having different compositions. These polymers were found to be soluble in aqueous buffers over wide pH ranges (ca 2-12). Further, we disclose methods involving the manipulation of polymer crosslinking conditions as functions of a number of factors such as monomer composition, pH, and the specific oxidizing agent utilized in the crosslinking reaction. The different oxidizing agents are shown to vary in their conversion of the catechol functionalities of the DOPA residues into o-quinone units, the units which are believed to be responsible for a variety of crosslinking reactions. J. H. Waite, Polym. Prepr., 30(1):181-182 (1990); J. H. Waite, Biol. Rev., 58:209-231 (1983). A illustration of an illustrative oxidizing agent reaction is provided in FIG. 3. By varying the conditions of the reaction environment, we illustrate systems where the characteristics of the polymer matrix are readily manipulated. In particular, by controlling the crosslinking reaction environment, gellation times could be adjusted from seconds to hours, and viscosity and adhesive strength could be controlled.